Novel inhibitors of dipeptidyl peptidase I

ABSTRACT

The present invention relates to new compounds that act as specific inhibitors of the cysteine protease dipeptidyl peptidase I (DP I). Those compounds are represented by the general formula  
                 
 
     and the pharmaceutical salts thereof, in which R is an acyl-residue including urethane and peptide, or a branched or unbranched C 1 -C 9  alkyl chain, a branched or unbranched C 2 -C 9  alkenyl chain, a branched or unbranched C 2 -C 9  alkynyl chain, a C 3 -C 9  cycloalkyl, C 4 -C 9  carbocyclic, C 5 -C 14  aryl, C 3 -C 9  heteroaryl, C 3 -C 9  heterocyclic, all of the above residues may be optionally substituted, or is H, AS is an amino acid or a peptide mimetic thereof. The amino acid is peptide bound with R and R′ is a branched or unbranched C 1 -C 9  alkyl chain, a branched or unbranched C 2 -C 9  alkenyl chain, a branched or unbranched C 2 -C 9  alkynyl chain, a C 3 -C 9  cycloalkyl, C 4 -C 9  cycloalkenyl, C 2 -C 9  heterocycloalkyl, C 3 -C 9  heterocycloalkenyl, C 5 -C 14  aryl, C 3 -C 9  heteroaryl, C 3 -C 9  heterocyclic, whereas the heterocycloalkyl, heterocycloalkenyl, heteroaryl, heterocyclic residue can have up to 6 hetero ring atoms, an amino acid or a peptide mimetic thereof, all of the above residues my be optionally substituted, or is H.

FIELD OF THE INVENTION

[0001] The present invention relates to compounds that act as specificinhibitors of the cysteine protease dipeptidyl peptidase I (DP I).Compounds based on acylated hydroxamates are distinguished by beingchemically stable in aqueous solutions, including biological fluids(Brömme & Demuth, 1994). Acylated hydroxamates are suicide inhibitorswhich deactivate the DP I by the reaction with the nucleophilic activesite thiol residue.

BACKGROUND OF INVENTION

[0002] Dipeptidyl peptidase I is known to release active granulocyteserine proteases of lymphatic cells from their pro-forms. Itparticipates in mechanisms that are used physiologically by cytotoxiclymphocytes in immune defence. In the case of pathophysiologicalprocesses such as malignant transformations of myeloid and lymphaticcells, the suppression of such mechanisms can be used for the treatmentof carcinomas, immune diseases or metabolic diseases. The inhibitors ofDP I according to the invention can be used for the treatment of suchpathophysiological conditions and diseases.

[0003] In addition to proteases involved in non-specific proteolysis,which ultimately results in the breakdown of proteins into amino acids,regulatory proteases are known which take part in the functionalisation(activation, deactivation, modulation) of endogenous peptides (Kirschkeet al., 1995; Krätusslich & Wimmer, 1987). In the immunological researchand neuropeptide research, a number of such so-called convertases,signal peptidases or enkephalinases have been discovered (Gomez et al.,1988; Ansorge et al., 1991).

[0004] Dipeptidyl peptidase I (DP I, Peptidase Classification Clan CA,Family C1, IUBMB Enzyme Classification EC 3.4.14.1, CAS Registration No.9032-68-2), formerly known as cathepsin C, was discovered in 1948 byGutman & Fruton. DP I removes dipeptides sequentially from unsubstitutedN-termini of polypeptide substrates with a relatively broad substratespecificity (McDonald et al., 1971; McDonald & Schwabe, 1977). DP I is alysosomal cysteine protease which, by removing N-terminal dipeptides, isable to release active enzymes from proenzymes, such as granzyme A,granzyme B, leucocyte elastase, cathepsin B, neuraminidase, in thelysosomal granula of cytotoxic T-lymphocytes (Kummer et al., 1996;Thiele & Lipsky, 1997).

[0005] Therefore It is commonly assumed that the DP I is involved inpathological mechanisms such as apoptotic processes, muscular dystrophyand carcinogenesis (Aoyagi et al., 1983; Gelman et al., 1980;Schlangenauff et al., 1992; Shi et al., 1992).

[0006] DP I is known as the convertase of the blood-sugar-raisinghormone glucagon which, in enzymatically reduced concentration, can leadto life-threatening hypoglycaemia (McDonald, J. K. et al., 1971).

[0007] Only weak inhibition of DP I is achieved by reversible andirreversible cysteine protease-inhibitors such as leupeptin and E-64,respectively (Nikawa et al., 1992). Stronger reversible inhibitors arestefin A and chicken cystatin, protein-inhibitors from the cystatinsuper-family, (Nicklin & Barrett, 1984). Specific inhibition has beenachieved with the a priori reactive affinity labels of the diazomethylketone and sulphonylmethyl ketone type (Angliker et al., 1989; Green &Shaw, 1981; Hanzlik, R. P. & Xing, R., 1998). In the last few years,other new reversible DP I-inhibitors and irreversibly acting affinitylabels of DP I have become known (Palmer et al., 1998; Thiele et al.,1997).

[0008] Such reversible inhibitors, which are able to display onlyshort-term effects caused by diffusion processes, and the affinitylabels that act irreversibly on the target enzyme in vitro but which,because of their chemically reactive radical which is present a priori,are able to react, prior to their interaction with the target enzyme,with other nucleophiles and electrophiles in biological fluids. Anothertype, mechanism-oriented inhibitors are distinguished by becomingcatalytically attacked and activated only by the target enzyme. Suchinhibitors are also known as suicide inactivators. Highly efficientsuicide inactivators for cysteine proteases have been developed with theclass of N-peptidyl, O-acyl hydroxylamines (Brömme et al., 1996).Inhibitors of DP I have not been derived from that class of compoundssince DP I is inert towards typical irreversible cysteineprotease-inhibitors known in the art, such as, for example, E-64.

[0009] Furthermore, N-terminally unprotected dipeptide derivatives tendtowards rapid, intramolecular decomposition.

[0010] Inhibitors of DP I are described in WO9324634; U.S. Pat. No.5,776,718; EP0995756; DE19834610; WO0220804; EP1188765, which areincorporated herein in their entirety concerning their structure,production and use.

[0011] Other helpful references include:

[0012] Ansorge, S., Schön, E., and Kunz, D. (1991). Membrane-boundpeptidases of lymphocytes: functional implications. Biomed.Biochim.Acta50, 799-807.

[0013] Angliker, H., Wikstrom, P., Kirschke, H., and Shaw, E. (1989).The inactivation of the cysteinyl exopeptidases cathepsin H and C byaffinity-labelling reagents. Biochem. J. 262, 63-68.

[0014] Aoyagi T., Wada, T., Kojima, F., Nagai, M., Miyoshino, S., andUmezawa, H. (1983). Two different modes of enzymatic changes in serumwith progression of Duchenne muscular dystrophy. Clin. Chim. Acta 129,165-173.

[0015] Brömme, D., Neumann, U., Kirschke, H., and Demuth, H. -U. (1996).Novel N-peptidyl-O-acyl hydroxamates: selective inhibitors of cysteineproteinases. Biochim. Biophys. Acta. 1202, 271-276.

[0016] Brömme,D., Demuth,H. U. (1994). N,O-Diacyl hydroxamates asselective and irreversible inhibitors of cysteine proteinases. Methodsin Enzym. 244, 671-685.

[0017] Gelman B. B., Papa, L., Davis, M. H., and Gruenstein, E. (1980).Decreased lysosomal dipeptidyl aminopeptidase I activity in culturedhuman skin fibroblasts in Duchenne's muscular dystrophy. J. Clin.Invest. 65, 1398-1406.

[0018] Gomez, S., Gluschankof, P., Lepage, A., and Cohen, P. (1988).Relationship between endo- and exopeptidases in a processing enzymesystem: activation of an endoprotease by the aminopeptidase B-likeactivity in somatostatin-28 convertase. Proc Natl Acad Sci USA 85,5468-5472.

[0019] Green G. D. J. & Shaw, E. (1981). Peptidyl diazomethyl ketonesare specific inactivators of thiol proteinases. J. Biol. Chem. 256,1923-1928.

[0020] Gutman H. R. & Fruton, J. S. (1948). On the proteolytic enzymesof animal tissues VIII. An intracellular enzyme related to chymotrypsin.J. Biol. Chem. 174, 851-858.

[0021] Hanzlik, R. P. & Xing, R. (1998). Azapeptides as inhibitors andactive site titrants for cysteine Proteinases. J. Med. Chem. 41,1344-1351.

[0022] Kirschke, H., Barrett, A. J., and Rawlings, N. D. (1995).Proteinases 1: lysosomal cysteine proteinases. Protein Profile 2,1581-1643.

[0023] Kräusslich, H. -G. and Wimmer, E. (1987). Viral Proteinases. Ann.Rev. Biochem. 57, 701

[0024] Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J. G.,and Hack, C. E. (1996). Expression of human recombinant granzyme Azymogen and its activation by the cysteine proteinase cathepsin C. J.Biol. Chem. 271, 9281-9286.

[0025] McDonald, J. K., Callahan, P. X., Ellis, S., and Smith, R. E.(1971). Polypeptide degradation by dipeptidyl aminopeptidase I(cathepsin C) and related peptidases. In: Tissue Proteinases (Barrett,A. J. & Dingle, J. T., eds). Amsterdam: North-Holland Publishing, pp.69-107.

[0026] McDonald, J. K. & Schwabe, C. (1977). Intracellularexopeptidases. In: Proteinases in mammalian cells and tissues (Barrett,A. J., ed.). Amsterdam: North Holland Publishing, pp. 311-391.

[0027] Nicklin, M. J. H. & Barrett, A. J. (1984). Inhibition of cysteineproteinases and dipeptidyl peptidase I by egg-white cystatin. Biochem.J. 223, 245-253.

[0028] Nikawa, T., Towatari, T., and Katunuma, N. (1992). Purificationand characterization of cathepsin J from rat liver. Eur. J. Biochem.204, 381-393.

[0029] Palmer, J. T., Rasnick, D., and Klaus, J. L. (1998). Reversibleprotease inhibitors. U.S. Pat. No. 5,776,718

[0030] Schlagenauff, B., Klessen, C., Teichmann-Dörr, S., Breuninger.H., and Rassner, G. (1992). Demonstration of proteases in basal cellcarcinomas. A histochemical study using aminoacid-4-methoxy-2-naphthylamides as chromogenic substrates. Cancer 70,1133-1140.

[0031] Shi, L., Kam, C. -M., Powers, J. C., Aebersold, R., andGreenberg, A. H. (1992). Purification of three cytotoxic lymphocytegranule serine proteases that induce apoptosis through distinctsubstrate and target cell interactions. J. Exp. Med. 176, 1521-1529.

[0032] Thiele, D. L., Lipsky, P. E., and McGuire, M. J. (1997).Dipeptidyl Peptidase-I inhibitors and uses thereof. U.S. Pat. No.5,602,102

SUMMARY OF THE INVENTION

[0033] The invention relates to inhibitors of DPI having the generalformula (I)

[0034] wherein

[0035] R is an acyl-residue including a urethane or peptide, or abranched or unbranched C₁-C₉ alkyl chain, a branched or unbranched C₂-C₉alkenyl chain, a branched or unbranched C₂-C₉ alkynyl chain, a C₃-C₉cycloalkyl, C₄-C₉ carbocyclic, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, all of the above residues optionally being substituted, orR is H, the residue AS—AS is a dipeptide or a mimetic thereof,

[0036] E is O or S, and

[0037] R′ is a branched or unbranched C₁-C₉ alkyl chain, a branched orunbranched C₂-C₉ alkenyl chain, a branched or unbranched C₂-C₉ alkynylchain, a C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl, C₂-C₉ heterocycloalkyl,C₃-C₉ heterocycloalkenyl, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, wherein the heterocycloalkyl, heterocycloalkenyl,heteroaryl, heterocyclic residue can have up to 6 hetero atoms in thering, or R′ is an amino acid or a peptide or a mimetic thereof, all ofthe above residues optionally being substituted, or R′ is H or alkoxy,alkenyloxy, alkynyloxy, carbocyclicoxy, heteroraryloxy, heterocyclicoxy,thioether or a substituted residue thereof or pharmaceuticallyacceptable salts thereof.

[0038] Examples of amino acids which can constitue the dipeptide AS—ASin the present invention are L and D-amino acids, N-methyl-amino-acids;allo- and threo-forms of Ile and Thr, which can, e.g. be α-, β- orω-amino acids, whereof α-amino acids are preferred.

[0039] R′ especially stands for H and for any alkyl, alkenyl, alkynyl,acyl, carbocyclic, aryl, heteroaryl, heterocyclic, alkoxy, alkenyloxy,alkynyloxy, carbocyclicoxy, heteroaryloxy, heterocyclicoxy, thioether ora substituted residue thereof.

[0040] The compounds according to the invention may also be in the formof prodrugs.

[0041] According to the invention, there are furthermore providedpharmaceutical compositions that comprise at least one compoundaccording to the invention, optionally in combination with carriersand/or adjuncts etc. that are customary per se.

[0042] The compounds and compositions according to the invention can beused for the in vivo inhibition of the enzyme dipeptidyl peptidase I orof enzymes similar to DP I.

[0043] They can be used especially for the treatment of diseases ofmammals that can be influenced by modulation of the DP I activity invarious cells, tissues and organs.

[0044] They are especially suitable for the treatment of DPI-mediatedmalignant cell degeneration, immune diseases and metabolic diseases ofhumans.

[0045] The present invention further relates to the use of the compoundsand compositions according to the invention for improving thewound-healing process and for the treatment of impaired wound-healing inhumans.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The invention relates to inhibitors of DP I having the generalformula (I):

[0047] wherein

[0048] R is an acyl-residue including a urethane or peptide, or abranched or unbranched C₁-C₉ alkyl chain, a branched or unbranched C₂-C₉alkenyl chain, a branched or unbranched C₂-C₉ alkynyl chain, a C₃-C₉cycloalkyl, C₄-C₉ carbocyclic, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, all of the above residues optionally being substituted, orR is H, the residue AS—AS is a dipeptide or a mimetic thereof,

[0049] E is O or S, and

[0050] R′ is a branched or unbranched C₁-C₉ alkyl chain, a branched orunbranched C₂-C₉ alkenyl chain, a branched or unbranched C₂-C₉ alkynylchain, a C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl, C₂-C₉ heterocycloalkyl,C₃-C₉ heterocycloalkenyl, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, wherein the heterocycloalkyl, heterocycloalkenyl,heteroaryl, heterocyclic residue can have up to 6 hetero atoms in thering, or R′ is an amino acid or a peptide or a mimetic thereof, all ofthe above residues optionally being substituted, or R′ is H or alkoxy,alkenyloxy, alkynyloxy, carbocyclicoxy, heteroraryloxy, heterocyclicoxy,thioether or a substituted residue thereof or pharmaceuticallyacceptable salts thereof.

[0051] Examples of amino acids which can be used in the presentinvention are L and D-amino acids, N-methyl-amino-acids; allo- andthreo-forms of Ile and Thr, which can, e.g. be α-, β- or ω-amino acids,whereof α-amino acids are preferred.

[0052] Preferably, the group AS—AS is bound with a peptide bond to R.

[0053] It has been found to be especially advantageous when the residueR is a phenyl or naphthyl residue that optionally is mono-, di-, orpoly-substituted by C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, acyl,C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, C₂-C₆ alkynyloxy, C₃-C₉ heteroaryloxy,C₃-C₉ heterocyclicoxy, C₁-C₆ thioether or a substituted residue thereof,NO₂, NH₂, F, Cl, Br, I atoms or groups. The above residues can bebranched or unbranched.

[0054] It is especially preferred when R′ is NO₂, NH₂, F, Cl, Br, Iatoms or groups or is a phenyl or naphthyl residue, which is optionallymono-, di-, or poly-substituted by C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ acyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, C₂-C₆ alkynyloxy,C₃-C₉ heteroaryloxy, C₃-C₉ heterocyclicoxy, C₁-C₆ thioether or asubstituted residue thereof, NO₂, NH₂, F, Cl, Br, I atoms or groups,

[0055] or

[0056] when R′ is

[0057] wherein V is N or CH and n=1-6

[0058] or pharmaceutically acceptable salts thereof.

[0059] In a further preferred embodiment, compounds of formula (I) areprovided,

[0060] wherein R′ is

[0061] wherein

[0062] T¹ is CH or N,

[0063] W¹, X¹, Y¹ and Z¹ are independently from each other selected fromCH₂, NR², N⁺(R³)₂, O, S, SO, S(R⁴)₂, and SO₂ with the proviso that atleast two or three of W¹, X¹, Y¹ and Z¹ are CH₂-groups, R², R³ and R⁴are independently from each other a branched or unbranched C₁-C₉ alkylchain, a branched or unbranched C₂-C₉ alkenyl chain, a branched orunbranched C₂-C₉ alkynyl chain, C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl orH or pharmaceutically acceptable salts thereof.

[0064] In another illustrative embodiment, compounds of formula (I) areprovided, wherein R′ is

[0065] wherein

[0066] T² is C or N⁺,

[0067] W², X², Y² and Z² are independently from each other CH, N, N⁺R⁵or S⁺R⁶ with the proviso that at least two or three of W², X², Y² and Z²are CH₂-groups,

[0068] R⁵ and R⁶ are independently from each other a branched orunbranched C₁-C₉ alkyl chain, a branched or unbranched C₂-C₉ alkenylchain, a branched or unbranched C₂-C₉ alkynyl chain, C₃-C₉ cycloalkyl,C₄-C₉ cycloalkenyl or H or pharmaceutically acceptable salts thereof.

[0069] Furthermore, compounds of formula (I) are provided, wherein R′ is

[0070] wherein

[0071] T³, W³, X³, Y³ and Z³ independently from each other are CH, N⁺R⁷or S⁺R⁸ with the proviso that at least two or three of T³, W³, X³, Y³and Z³ are CH₂-groups,

[0072] R⁷ and R⁸ are independently a branched or unbranched C₁-C₉ alkylchain, a branched or unbranched C₂-C₉ alkenyl chain, a branched orunbranched C₂-C₉ alkynyl chain, C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl orH,

[0073] or pharmaceutically acceptable salts thereof.

[0074] The present invention further provides compounds of formula (I),wherein R′ is

[0075] wherein

[0076] T⁴ is C or N⁺, or pharmaceutically acceptable salts thereof.

[0077] The residues R′ defined in the description and the claims may bemono- or poly-substituted by, e.g., alkyl, alkoxy, alkenyl, alkynyl,acyl, carbocyclic, aryl, heteroaryl, heterocyclic, thioether, NO₂, NH₂,F, Cl, Br, I atoms or groups, mono- or di-substitution being preferred.It is especially preferred that the substituents are not substituted anyfurther.

[0078] Furthermore the present invention provides compounds of formula(I), wherein R′ is an amino acid, a peptide or a dipeptide or a mimeticthereof.

[0079] The salts of the compounds of the invention may, assuming thatthey have basic properties, be in the form of inorganic or organicsalts.

[0080] The compounds of the present invention can be converted into andused as acid addition salts, especially pharmaceutically acceptable acidaddition salts. The pharmaceutically acceptable salt generally takes aform in which a basic side chain is protonated with an inorganic ororganic acid. Representative organic or inorganic acids includehydrochloric, hydrobromic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic,tartaric, citric, benzoic, mandelic, methanesulfonic,hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toulenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic acid. All pharmaceuticallyacceptable acid addition salt forms of the compounds of the presentinvention are intended to be embraced by the scope of this invention.

[0081] In view of the close relationship between the free compounds andthe compounds in the form of their salts, whenever a compound isreferred to in this context, a corresponding salt is also intended,provided such is possible or appropriate under the circumstances.

[0082] Where the compounds according to this invention have at least onechiral center, they may accordingly exist as enantiomers. Where thecompounds possess two or more chiral centers, they may additionallyexist as diastereomers. It is to be understood that all such isomers andmixtures thereof are encompassed within the scope of the presentinvention. Furthermore, some of the crystalline forms of the compoundsmay exist as polymorphs and as such are intended to be included in thepresent invention. In addition, some of the compounds may form solvateswith water (i.e. hydrates) or common organic solvents, and such solvatesare also intended to be encompassed within the scope of this invention.

[0083] The compounds, including their salts, can also be obtained in theform of their hydrates, or include other solvents used for theircrystallization.

[0084] Compounds comprising acylated hydroxamates are new inhibitorystructures. With their selectivity and stability they fulfil therequirements for the development of new drugs.

[0085] Throughout the description and the claims the expression “acyl”can denote a C₁₋₂₀ acyl residue, preferably a C₁₋₈ acyl residue andespecially preferred a C₁₋₄ acyl residue, “carbocyclic” or “cycloalkyl”can denote a C₃₋₁₂ carbocyclic residue, preferably a C₄, C₅ or C₆carbocyclic residue, “cycloalkenyl” can denote a C₃₋₁₂ carbocyclicresidue, preferably a C₅ or C₆ carbocyclic residue having at least onedouble band at any desired location. “Heteroaryl” is defined as an arylresidue, wherein 1 to 4, preferably 1, 2 or 3 ring atoms are replaced byheteroatoms like N, S or O. “Heterocycloalkyl” or “heterocyclic” isdefined as a cycloalkyl residue, wherein 1, 2 or 3 ring atoms arereplaced by heteroatoms like N, S or O. “Heterocycloalkenyl” is definedas a heterocycloalkyl residue having at least one double bond at anydesired location. The expression “alkyl” can denote a C₁₋₅₀ alkyl group,preferably a C₆₋₃₀ alkyl group, especially a C₈₋₁₂ alkyl group; an alkylgroup may also be a methyl, ethyl, propyl, isopropyl or butyl group. Theexpression “aryl” is defined as an aromatic residue, preferablysubstituted or optionally unsubstituted phenyl, benzyl, naphthyl,biphenyl or anthracene groups, which preferably have 6-24, morepreferred 8-14 C ring atoms; the expression “alkenyl” can denote a C₂₋₁₀alkenyl group, preferably a C₂₋₆ alkenyl group, which has the doublebond or the double bonds at any desired location and may be substitutedor unsubstituted; the expression “alkynyl” can denote a C₂₋₁₀ alkynylgroup, preferably a C₂₋₆ alkynyl group, which has the triple bond or thetriple bonds at any desired location and may be substituted orunsubstituted; the expression “alkoxy” can denote a C₁₋₅₀ alkyl-oxygengroup, preferably a C₁₋₆ alkyl-oxygen group; the expression “alkenyloxy”can denote a C₂₋₁₀ alkenyl-oxygen group, preferably a C₂₋₆alkenyl-oxygen group; the expression “alkynyloxy” can denote a C₂₋₁₀alkynyl-oxygen group, preferably a C₂₋₆ alkynyl-oxygen group; theexpression “carbocyclicoxy” can denote a C₃₋₁₂ carbocyclic-oxygen group;the expression “heteroaryloxy” can denote a heteroaryl-oxygen group, theexpression “heterocyclicoxy” can denote a heterocyclic-oxygen group; theexpression “substituted” can denote any desired substitution by one ormore, preferably one or two, alkyl, alkenyl, alkynyl, mono- ormulti-valent acyl, alkoxy, alkoxyacyl, alkenyloxy, alkynyloxy,carbocyclicoxy, heteroaryloxy, heterocyclicoxy, alkoxyalkyl groups, anymonoether or polyether containing identical or different alkyl, aryl,alkenyl, alkynyl, carbocyclic, heteroaryl, heterocyclic residues, or anymonothioether or polythioether containing identical or different alkyl,aryl, alkenyl, alkynyl, carbocyclic, heteroaryl, heterocyclic residues;the afore-mentioned substituents may in turn have one or more (butpreferably zero) alkyl, alkenyl, alkynyl, mono- or multi-valent acyl,alkoxyacyl or alkoxyalkyl groups as side groups which are preferably notsubstituted themselves. Organic amines, amides, alcohols or acids, eachhaving from 8 to 50 C atoms, preferably from 10 to 20 C atoms, can havethe formulae (alkyl)₂N— or alkyl-NH—, —CO—N(alkyl)₂ or —CO—NH(alkyl),-alkyl-OH or -alkyl-COOH.

[0086] The expression urethanes can denote a compound of the formulaR—NH—CO—OR″″, wherein R″″ is a substituted alkyl, acyl, alkenyl,alkynyl, carbocyclic, heteroaryl, heterocyclic or aryl residues. R isidentical to the residue R of formula I and is as defined for formula I.Preferred for R″″ are unsubstituted or substituted alkyl residues, e. g.methyl, ethyl, tert-butyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl; unsubstituted or substituted alkenyl residues,e.g. allyl; unsubstituted or substituted aryl residues, e.g. phenyl,benzyl, 9-fluorenylmethyl.

[0087] All of the above residues or groups can—if possible—be branchedor unbranched, unsubstituted or substituted with, e.g., 1, 2, 3, 4 or 5substitutents, whereof 1 or 2 substituents are preferred.

[0088] The expression “peptide” for the definition of the residue R candenote any di-, tri-, tetra-, penta-, hexa-, or polypeptide. The peptidecan be constituted of any amino acids or mimetics of amino acids orpeptides.

[0089] The group AS—AS can be constituted of any two amino acids ormimetics thereof.

[0090] Examples of amino acids are:

[0091] aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), lysine(Lys), histidine (His), glycine (Gly), serine (Ser) and cysteine (Cys),threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr),alanine (Ala), proline (Pro), valine (Val), isoleucine (Ile), leucine(Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp),hydroxyproline (Hyp), beta-alanine (beta-Ala), 2-amino octanoic acid(Aoa), azetidine-(2)-carboxylic acid (Ace), pipecolic acid (Pip),3-amino propionic, 4-amino butyric and so forth, alpha-aminoisobutyricacid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit),homoarginine (Har), t-butylalanine (t-butyl-Ala), t-butylglycine(t-butyl-Gly), N-methylisoleucine (N-Melle), phenylglycine (Phg),cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) andmethionine sulfoxide (MSO), Acetyl-Lys, modified amino acids such asphosphoryl-serine (Ser(P)), benzyl-serine (Ser(Bzl)) andphosphoryl-tyrosine (Tyr(P)), 2-aminobutyric acid (Abu),aminoethylcysteine (AECys), carboxymethylcysteine (Cmc), dehydroalanine(Dha), dehydroamino-2-butyric acid (Dhb), carboxyglutaminic acid (Gla),homoserine (Hse), hydroxylysine (Hyl), cis-hydroxyproline (cisHyp),trans-hydroxyproline (transHyp), isovaline (Iva), pyroglutamic acid(Pyr), norvaline (Nva), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid(3-Abz), 4-aminobenzoic acid (4-Abz), 4-(aminomethyl)benzoic acid (Amb),4-(aminomethyl)cyclohexanecarboxylic acid (4-Amc), Penicillamine (Pen),2-Amino-4-cyanobutyric acid (Cba), cycloalkane-carboxylic acids.Examples of {overscore (ω)}-amino acids are e.g.: 5-Ara (aminoralericacid), 6-Ahx (aminohexanoic acid), 8-Aoc (aminooctanoic acid), 9-Anc(aminovanoic acid), 10-Adc (aminodecanoic acid), 11-Aun (aminoundecanoicacid), 12-Ado (aminododecanoic acid). Further amino acids are:indanylglycine (Igl), indoline-2-carboxylic acid (Idc),octahydroindole-2-carboxylic acid (Oic), diaminopropionic acid (Dpr),diaminobutyric acid (Dbu), naphtylalanine (1-Nal), (2-Nal),4-aminophenylalanin (Phe(4-NH₂)), 4-benzoylphenylalanine (Bpa),diphenylalanine (Dip), 4-bromophenylalanine (Phe(4-Br)),2-chlorophenylalanine (Phe(2-Cl)), 3-chlorophenylalanine (Phe(3-Cl)),4-chlorophenylalanine (Phe(4-Cl)), 3,4-chlorophenylalanine(Phe(3,4-Cl₂)), 3-fluorophenylalanine (Phe(3-F)), 4-fluorophenylalanine(Phe(4-F)), 3,4-fluorophenylalanine (Phe(3,4-F₂)),pentafluorophenylalanine (Phe(F₅)), 4-guanidinophenylalanine(Phe(4-guanidino)), homophenylalanine (hPhe), 3-jodophenylalanine(Phe(3-J)), 4-jodophenylalanine (Phe(4-J)), 4-methylphenylalanine(Phe(4-Me)), 4-nitrophenylalanine (Phe-4-NO₂)), biphenylalanine (Bip),4-phosphonomehtylphenylalanine (Pmp), cyclohexyglycine (Ghg),3-pyridinylalanine (3-Pal), 4-pyridinylalanine (4-Pal),3,4-dehydroproline (A-Pro), 4-ketoproline (Pro(4-keto)), thioproline(Thz), isonipecotic acid (Inp),1,2,3,4,-tetrahydroisoquinolin-3-carboxylic acid (Tic), propargylglycine(Pra), 6-hydroxynorleucine (NU(6-OH)), homotyrosine (hTyr),3-jodotyrosine (Tyr(3-J)), 3,5-dijodotyrosine (Tyr(3,5-J₂)),d-methyl-tyrosine (Tyr(Me)), 3-NO₂-tyrosine (Tyr(3-NO₂)),phosphotyrosine (Tyr(PO₃H₂)), alkylglycine, 1-aminoindane-1-carboxyacid, 2-aminoindane-2-carboxy acid (Aic),4-amino-methylpyrrol-2-carboxylic acid (Py),4-amino-pyrrolidine-2-carboxylic acid (Abpc),2-aminotetraline-2-carboxylic acid (Atc), diaminoacetic acid (Gly(NH₂)),diaminobutyric acid (Dab), 1,3-dihydro-2H-isoinole-carboxylic acid(Disc), homocylcohexylalanin (hCha), homophenylalanin (hPhe oder Hof),trans-3-phenyl-azetidine-2-carboxylic acid,4-phenyl-pyrrolidine-2-carboxylic acid,5-phenyl-pyrrolidine-2-carboxylic acid, 3-pyridylalanine (3-Pya),4-pyridylalanine (4-Pya), styrylalanine,tetrahydroisoquinoline-1-carboxylic acid (Tiq),1,2,3,4-tetrahydronorharmane-3-carboxylic acid (Tpi),β-(2-thienyl)-alanine (Tha).

[0092] Proteinogenic amino acids are defined as natural protein-derived(α-amino acids. Non-proteinogenic amino acids are defined as all otheramino acids, which are not building blocks of common natural proteins.

[0093] Peptide mimetics per se are known to a person skilled in the art.They are preferably defined as compounds which have a secondarystructure like a peptide and optionally further structuralcharacteristics; their mode of action is largely similar or identical tothe mode of action of the native peptide; however, their activity (e.g.as an antagonist or inhibitor) can be modified as compared with thenative peptide, especially vis à vis receptors or enzymes. Moreover,they can imitate the effect of the native peptide (agonist). Examples ofpeptide mimetics are scaffold mimetics, non-peptidic mimetics,peptoides, peptide nucleic acids, oligopyrrolinones, vinylogpeptides andoligocarbamates. For the definitions of these peptide mimetics seeLexikon der Chemie, Spektrum Akademischer Verlag Heidelberg, Berlin,1999.

[0094] The aim for using these mimetic structures is increasing theactivity, increasing the selectivity to decrease side effects, protectthe compound (drug) against enzymatic degradation for prolongation ofthe effect.

[0095] Further peptide mimetics are defined in J. Gante, Angew. Chemie,1994, 106, 1780-1802; V. J. Hruby et al., Biopolymers, 1997, 219-266; D.Nöteberg et al., 2000, 43, 1705-1713.

[0096] The present invention further includes within its scope prodrugsof the compounds, provided herein. In general, such prodrugs will befunctional derivatives of the compounds which are readily convertible invivo into the desired therapeutically active compound. Thus, in thesecases, the use of the present invention shall encompass the treatment ofthe various disorders described with prodrug versions of one or more ofthe claimed compounds, but which converts to the above specifiedcompound in vivo after administration to the subject. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs”, ed. H.Bundgaard, Elsevier, 1985.

[0097] Such prodrugs can be cleaved and the active inhibitors can bereleased. This activation of the active inhibitors can be achieved bothby chemical and enzymatic reactions. Esterases, proteases and peptidasesserve to release the active inhibitors from the compounds according tothe invention. Esterases, proteases and peptidases, which are suitablein such manner, are disclosed in WO 97/45117, U.S. Pat. No. 5,433,955,U.S. Pat. No. 5,614,379 and U.S. Pat. No. 5,624,894. Preferred proteasesare aminopeptidases, dipeptidyl aminopeptidases, endoproteases, andendopeptidases. Especially preferred proteases for the release of theactive inhibitors from the precursor of the present invention areaminopeptidase N, aminopeptidase P, pyroglutaminyl aminopeptidase,dipeptidyl peptidase IV and dipeptidyl peptidase IV-like enzymes. Suchproteases and their specificity are described in:

[0098] Handbook of Proteolytic Enzymes, Eds. Barrett, A. J., Rawlings,N. D. and Woessner, J. F. Academic Press, New York 1998.

[0099] The present invention accordingly also uses the concept tostabilize e.g. unstable inhibitors by masking them in prodrug form.Thereby the properties of the active inhibitors can be modulated. Forexample, the prodrugs according to the invention have the advantage thatthe active inhibitors of DP I are released according to individualpatients′ needs. Moreover, this invention has the further advantage thateach organism will release exactly that amount of active inhibitor thatis necessary to inhibit that amount of DP I molecules, which is presentin the body of the respective organism.

[0100] When a prodrug according to the invention interacts with anenzyme as mentioned above, it is cleaved by this enzyme and the activeinhibitor is released. The active inhibitor will inhibit DP I so that DPI cannot cleave any further compounds for a defined time. In certaincases, e.g. when the enzyme cleaving the prodrugs is DP I, the remainingprodrugs are not degraded during a defined time and thus, constitute aninhibitor reservoir until the concentration of DP I molecules risesagain or active inhibitor molecules are eliminated or inactivated.

[0101] To summarise, it may be stated that, using the prodrugs of thepresent invention, it is possible in a completely surprising manner:

[0102] 1. to achieve increased action of the inhibitors;

[0103] 2. to release the active inhibitors according to the patient'sneeds;

[0104] 3. to release the active inhibitors in a temporally controlledmanner;

[0105] 4. to release the active inhibitors in a site-specific manner;and

[0106] 5. to provide a reservoir of DP I inhibitors.

[0107] According to the invention, there are furthermore providedpharmaceutical compositions that comprise at least one compoundaccording to the invention, optionally in combination with carriersand/or adjuncts etc. that are customary per se.

[0108] The compounds and compositions according to the invention can beused for the in vivo inhibition of the enzyme dipeptidyl peptidase I orof enzymes similar to DP I.

[0109] They can be used especially for the treatment of diseases ofmammals that can be influenced by modulation of the DP I activity invarious cells, tissues and organs.

[0110] They are especially suitable for the treatment of DP I-mediatedmalignant cell degeneration, immune diseases and metabolic diseases ofhumans.

[0111] The present invention further relates to the use of the compoundsand compositions according to the invention for improving thewound-healing process and for the treatment of impaired wound-healing inhumans.

[0112] The compounds may especially be in prodrug form and be used inprodrug form.

[0113] Biological Evaluation

[0114] The compounds provided herein are highly specific inhibitors ofthe cysteine protease DP I. Selected compounds, based on the generalformula (II) below, were tested concerning their inhibitory potentialagainst DP I and for their cross-reactivity against three more cysteineproteases, namely cathepsin B, H, L and another dipeptidyl peptidase,dipeptidyl peptidase IV (DP IV). The IC₅₀ values of the tested compoundsare given in table 1. The inhibitory potential of the compounds is shownin table 2 (k_(inact)/K_(i)). TABLE 1 IC₅₀ values of the compounds offormula II formula II

IC₅₀ [μM] compound R R′ DP I Cath. B Cath. H Cath. L DP IV 10 H CH₃0.258 117 n.i. 156 n.i. 11 H C₆H₅ 0.034 37.7 n.i. 36.9 n.i. 12 HC₆H₅-p-CH₃ 0.203 34.05 154 33.05 n.i. 13 H C₆H₅-p-NO₂ 0.00262 5.31 34.77.32 ni. 14 CH₃ C₆H₅ 0.157 41.8 205 4.61 n.i.

[0115] Cytotoxicity Evaluation

[0116] The toxicological potential of the compounds provided thereinagainst murine cell lines is different from their toxicologicalpotential against human cell lines. Selected compounds, based on thegeneral formula (II) above, were tested concerning theircytotoxicological potential against the two cell-lines, L-929 (murinefibroblast cell line) and Hep-G2 (human hepatocyte cell line) cells. TheLD₅₀ values of the tested compounds are given in table 2. TABLE 2 LD₅₀values of the compounds k_(inact)/K_(i) LD₅₀ [mg*ml⁻¹] compound [M⁻¹s⁻¹]L-929 Hep-G2 10 3.9 * 10⁴ 4.3 1.6 11   4 * 10⁵ 1.0 0.8 12 1.9 * 10⁴ n.d.n.d. 13 7.9 * 10⁴ 2.3 0.8 14 1.4 * 10⁵ n.d. n.d.

[0117] Synthesis

[0118] The inhibitors 10-14 were prepared as described in Scheme 1. Thedipeptides 1 and 2 were prepared starting from HCl*H-Phe-OMe andBoc-Gly-OH or Boc-Sar-OH respectively (obtained from Bachem) accordingto a procedure described in Bodansky, M. and Bodansky,A. (Method A). Thedipeptides were converted into the peptidylhydroxylamines 3 and 4 bytreatment with hydroxylamine (Method B). Acylation with various carbonicacid chlorides in the presence of triethylamine gave the correspondingacetyl derivatives 5-9 (Method C). Treatment of the compounds 5-9 withtrifluoroacetic acid provided the inhibitors 10-14 (Method D).

[0119] Experimental

[0120] NMR spectra were performed on Varian Unity 500 and Bruker AM 400spectrometers. The following abbrevations are used: s, singlet; d,doublet; t, triplet; q, quartet; br., broad. Melting points weremeasured on a Leica Galen III melting point apparatus and areuncorrected. ESI-MS: Mass spectra taken with an MDS Sciex API 365 massspectrometer equipped with an Ionspray™ interface (MDS Sciex; ThornHill, ON, Canada). The instrument settings, data acquisition andprocessing were controlled by the Applied Biosystems (Foster City,Calif., USA) Analyst™ software for Windows NT™. 50-100 scans wereperformed by the positive ionization Q1 scan mode to accumulate thepeaks. Sample solutions were diluted with 50% methanol in 0.5% formicacid to reach concentrations about 10 μg/ml. Each sample solution wasintroduced directly by a microsyringe (1 ml) through an infusion pump(Havard Apperatus 22; Havard Instruments; Holliston, Mass., USA) andfused silica capillary tubing at a rate of 20 μl/min. Thin layerchromatography (TLC) was done with Macherey Nagel Polygram® SIL G/UV₂₄₅.Visualisation was accomplished by means of UV ligth at 254 nm, followedby dyeing with potassium permanganate or ninhydrin. Solvents weredistilled prior to use. Petroleum ether with a boiling range of 35-65°C. was used. All commercially available reagents were used withoutfurther purification. Amino acid derivates were obtained from Bachem.For the purification a preparative HPLC [acetonitrile-water, gradient:5-95%, flow rate: 6 ml min⁻¹, column: Nucleosil 7μ C18 100A, 250×21.2 mm(phenomenex), pump: L-6250 Merck-Hitachi] was used.

[0121] Abbrevations

[0122] Ac: Acetyl (COCH₃), Bz: Benzoyl (COC₆H₅), CAIBE: Isobutylchloroformate, NMM: N-Methylmorpholine, PE: Petroleum ether.

[0123] General Methods

[0124] Method A (Preparation of the dipeptides): HCl*H-L-Phe-OMe andBoc-Gly-OH or Boc-Sar-OH were coupled according to the method ofBodansky, M. and Bodansky,A., The Practice of Peptide Synthesis 2^(nd)Edition, Springer-Verlag. To a stirred solution of Boc-Gly-OH orBoc-Sar-OH (17.1 mmol, 1.0 equiv) in 75 ml dry THF was added NMM (1.88ml, 17.1 mmol, 1.0 equiv). After cooling the mixture to −15° C. CAIBE(2.22 ml, 17.1 mmol, 1.0 equiv) was added and after stirring for further15 min HCl*H-L-Phe-OMe (3.69 g, 17.1 mmol, 1.0 equiv) and NMM (1.88 ml,17.1 mmol, 1.0 equiv) were added. The mixture was stirred for 14 h,during which time it was allowed to warm to room temperature. Thesolvent was evaporated in vacuo and the obtained residue was dissolvedin ethyl acetate (50 ml), washed with 1 N HCl, water, aqueous NaHCO₃,and brine (30 ml per washing step). The organic layer was dried overNa₂SO₄, filtered and concentrated under reduced pressure. The productwas crystallized by means of ethyl acetate/pentane and used withoutfurther characterization.

[0125] Method B (Conversation into the peptidylhydroxylamines): Thepeptidylhydroxylamines were prepared according to the method of Brömme,D., Demuth, H. U., N,O-Diacyl hydroxamates as selective and irreversibleinhibitors of cysteine proteinases. Methods in Enzym. 244, 671-685.Hydroxylamine hydrochloride (827 mg, 11.9 mmol, 4.0 equiv) was dissolvedin 18 ml of dry methanol. 17 ml of fresh prepared NaOMe solution (3.5 Min absolute methanol) was added dropwise. The mixture was filtered after20 min of stirring and the filtrate was dropped into a chilled andstirred solution of the dipeptide (2.97 mmol, 1.0 equiv) in 4 ml of drymethanol. After 8 h of stirring at 0° C. the solvent was removed and theremaining oil was taken up in 3 ml of water and extracted with 3 ml ofethyl acetate. The aqueous phase was brought to a pH-value of 3 by meansof adding KHSO₄ and again extracted three times by means of 15 ml ethylacetate. The organic layer was dried over Na₂SO₄, filtered andevaporated. The residue was recrystallized by means of MeOH/pentane.

[0126] Method C (Acylation): To a stirred solution of apeptidylhydroxylamine (2 mmol, 1.0 equiv) in a mixture of dry THF (5 ml)and dry DMF (3 ml) at −15° C. was added triethylamine (20.0 μl, 2.2mmol, 1.1 equiv) and the corresponding carbonic acid chlorides (2.1mmol, 1.05 equiv) dissolved in dry THF (4 ml). This solution was stirredfor 4 hours at −5° C. before the solvents were removed under reducedpressure. The obtained residue was washed with cold KHSO₄ (5% in water,5 ml). The precipitate was dissolved in ethyl acetate (10 ml) and driedover Na₂SO₄. After filtration the solvent was evaporated under reducedpressure and the remaining residue was recrystallized by means ofMeOH/PE.

[0127] Method D (Deprotecting the boc protecting group): TheBoc-protected compound (2 mmol) was dissolved in trifluoroacetic acid(10 ml) and the solution was stirred for 1.5 h at room temperaturebefore it was diluted with toluol (3 ml). The solvents were removedunder reduced pressure and the obtained residue was triturated with Et₂O(10 ml) and filtered. The resulted solid was washed three times withEt₂O (5 ml) and dried. The obtained residue was purified by flashchromatography to give the desired compound.

[0128] Starting Material

Synthesis of Boc-Gly-L-Phe-OMe (1)

[0129] Boc-Gly-L-Phe-OMe (1) was prepared according to Method A in ayield of 98%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.71.—¹H NMR (500 MHz,DMSO-d₆): δ=1.36 (s, 9 H, t-Bu), 2.90 (dd, 1 H, J=13.7 Hz, J=8.7 Hz, CH₂Phe), 3.00 (dd, 1 H, J=13.7 Hz, J=8.7 Hz, CH₂ Phe), 3.58 (s, 3 H, OCH₃),4.01 (q, 2 H, J=7.1 Hz, CH₂ Gly), 4.46 (dd, 1 H, J=13.7 Hz, J=8.1 Hz, CHGly), 6.90 (t, 1 H, J=6.0 Hz, NH), 7.17-7.22 (m, 3 H, aryl-H), 7.25-7.28(m, 2 H, aryl-H), 8.20 (d, 1 H, J=7.6 Hz, NH).—MS (EI) m/z (%): 337[M+H⁺], 354 [M+NH₄ ⁺], 359 [M+Na⁺], 375 [M+K⁺].

Synthesis of Boc-Sar-L-Phe-OMe (2)

[0130] Boc-Sar-L-Phe-OMe (2) was prepared according to Method A in ayield of 99%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.82.—MS (EI) m/z (%): 351[M+H⁺], 368 [M+NH₄ ⁺], 373 [M+Na⁺], 389 [M+K⁺].

Synthesis of Boc-Gly-L-Phe-NHOH (3)

[0131] Boc-Gly-L-Phe-NHOH (3) was prepared according to Method B in ayield of 72%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.44.—¹H NMR (500 MHz,DMSO-d₆): δ=1.36 (s, 9 H, t-Bu), 277 (dd, 1 H, J=13.7 Hz, J=8.9 Hz, CH₂Phe), 2.91 (dd, 1 H, J=13.7 Hz, J=8.3 Hz, CH₂ PHe), 341 (dd, 1 H, J=16.7Hz, J=6.2 Hz, CH₂ Gly), 3.53 (dd, 1 H, J=16.7 Hz, J=6.2 Hz, CH₂ Gly),4.34-4.38 (m, 1 H, CH Phe), 6.86 (t, 1 H, J=6.1 Hz, NH), 7.16-7.19 (m, 3H, aryl-H), 7.23-7.28 (m, 2 H, aryl-H), 8.06 (d, 1 H, J=8.5 Hz, NH),8.88 (s, 1 H, OH), 10.65 (s, 1 H, NH).—MS (EI) m/z (%): 338 [M+H⁺], 355[M+NH₄ ⁺], 360 [M+Na⁺], 376 [M+K⁺].

Synthesis of Boc-Sar-L-Phe-NHOH (4)

[0132] Boc-Sar-L-Phe-NHOH (4) was prepared according to Method B in ayield of 88%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.35.—MS (EI) m/z (%): 352[M+H⁺], 369 [M+NH₄ ⁺], 374 [M+Na⁺], 390 [M+K⁺].

Synthesis of Boc-Gly-L-Phe-NHO-Ac (5)

[0133] Boc-Gly-L-Phe-NHO-Ac (5) was prepared according to Method C in ayield of 62%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.69.—¹H NMR (500 MHz,DMSO-d₆): δ=1.35 (s, 9 H, t-Bu), 2.14 (s, 3 H, CH₃), 2.80 (dd, 1 H,J=13.7 Hz, J=9.6 Hz, CH₂ Phe), 2.98 (dd, 1 H, J=13.7 Hz, J=9.8 Hz, CH₂Phe), 3.42 (dd, 1 H, J=16.9 Hz, J=6.2 Hz, CH₂ Gly), 3.54 (dd, 1 H,J=16.9 Hz, J=6.2 Hz, CH₂ Gly), 4.50-4.54 (m, 1 H, CH Phe), 6.85 (t, 1 H,J=6.1 Hz, NH), 7.17-7.27 (m, 5 H, aryl-H), 8.18 (d, 1 H, J=8.4 Hz, NH),12.00 (s, 1 H, NH).—MS (EI) m/z (%): 380 [M+H⁺], 397 [M+NH₄ ⁺], 402[M+Na⁺], 418 [M+K⁺].

Synthesis of Boc-Gly-L-Phe-NHO-Bz (6)

[0134] Boc-Gly-L-Phe-NHO-Bz (6) was prepared according to Method C in ayield of 48%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.66.—MS (EI) m/z (%): 442[M+H⁺], 459 [M+NH₄ ⁺], 464 [M+Na⁺], 480 [M+K⁺].

Synthesis of Boc-Gly-L-Phe-NHO-Bz-p-CH₃ (7)

[0135] Boc-Gly-L-Phe-NHO-Bz-p-CH₃ (7) was prepared according to Method Cin a yield of 52%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.55.—MS (EI) m/z (%):456 [M+H⁺], 473 [M+NH₄ ⁺], 478 [M+Na⁺], 494 [M+K⁺].

Synthesis of Boc-Gly-L-Phe-NHO-Bz-p-NO₂ (8)

[0136] Boc-Gly-L-Phe-NHO-Bz-p-NO₂ (8) was prepared according to Method Cin a yield of 62%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.57.—MS (EI) m/z (%):487 [M+H⁺], 504 [M+NH₄ ⁺], 509 [M+Na⁺], 525 [M+K⁺].

Synthesis of Boc-Sar-L-Phe-NHO-Bz (9)

[0137] Boc-Sar-L-Phe-NHO-Bz (9) was prepared according to Method C in ayield of 78%.—TLC (MeOH/CHCl₃, 1:9): R_(f)=0.62.—MS (EI) m/z (%): 456[M+H⁺], 473 [M+NH₄ ⁺], 478 [M+Na⁺], 494 [M+K⁺].

[0138] Pharmaceutical Compositions

[0139] Additionally, the present invention includes the use of thecompounds provided herein for the preparation of a medicament for thetreatment of a condition mediated by modulation of the DP I activity ina subject. The compound may be administered to a patient by anyconventional route of administration, including, but not limited to,intravenous, oral, subcutaneous, intramuscular, intradermal, parenteraland topical. Especially preferred is topical administration.

[0140] The present invention also provides pharmaceutical compositionscomprising one or more compounds of this invention in association with apharmaceutically active carrier.

[0141] To prepare the pharmaceutical compositions of this invention, oneor more active compounds or salts thereof of the invention as the activeingredient, is intimately admixed with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques, whichcarrier may take a wide variety of forms depending of the form ofpreparation desired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like; for solidoral preparations such as, for example, powders, capsules, gelcaps andtablets, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like. Because of their ease in administration, tablets andcapsules represent the most advantageous oral dosage unit form, in whichcase solid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar coated or enteric coated by standard techniques.For parenterals, the carrier will usually comprise sterile water,through other ingredients, for example, for purposes such as aidingsolubility or for preservation, may be included. For topicalformulations, such as creams, gels etc., the carrier will usuallycomprise glycerolmonostearate, cetyl alcohol, triglycerides, vaseline,propylenglycole, water and paraffins.

[0142] Injectable suspensions may also prepared, in which caseappropriate liquid carriers, suspending agents and the like may beemployed. The pharmaceutical compositions herein will contain, perdosage unit, e.g., tablet, capsule, powder, injection, teaspoonful andthe like, an amount of the active ingredient necessary to deliver aneffective dose as described above. The pharmaceutical compositionsherein will contain, per unit dosage unit, e.g., tablet, capsule,powder, injection, suppository, teaspoonful and the like, of from about0.03 mg to 100 mg/kg/bw (preferred 0.1-30 mg/kg/bw) and may be given ata dosage of from about 0.1-300 mg/kg/day/bw (preferred 1-50mg/kg/day/bw). The dosages, however, may be varied depending upon therequirement of the patients, the severity of the condition being treatedand the compound being employed. The use of either daily administrationor post-periodic dosing may be employed.

[0143] Preferably these compositions are in unit dosage forms from suchas tablets, pills, capsules, powders, granules, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampoules, autoinjector devices or suppositories; for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. Alternatively, the composition may bepresented in a form suitable for once-weekly or once-monthlyadministration; for example, an insoluble salt of the active compound,such as the decanoate salt, may be adapted to provide a depotpreparation for intramuscular injection. For preparing solidcompositions such as tablets, the principal active ingredient is mixedwith a pharmaceutical carrier, e.g. conventional tableting ingredientssuch as corn starch, lactose, sucrose, sorbitol, talc, stearic acid,magnesium stearate, dicalcium phosphate or gums, and otherpharmaceutical diluents, e.g. water, to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective dosage forms such as tablets, pills and capsules. Thissolid preformulation composition is then subdivided into unit dosageforms of the type described above containing from 0.01 to about 500 mgof the active ingredient of the present invention.

[0144] The tablets or pills of the novel composition can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermits the inner component to pass intact into the duodenum or to bedelayed in release. A variety of material can be used for such entericlayers or coatings, such materials including a number of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate.

[0145] This liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions, include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

[0146] Most preferred according to this invention are formulations fortopical administration of the compounds provided herein, such as creams,gels and sprays. Suitable carriers for topical administration includeglycerolmonostearate, cetyl alcohol, triglycerides, vaseline,propylenglycole, water or paraffins.

[0147] Where the processes for the preparation of the compoundsaccording to the invention give rise to mixture of stereoisomers, theseisomers may be separated by conventional techniques such as preparativechromatography. The compounds may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their components enantiomers by standard techniques, such as theformation of diastereomeric pairs by salt formation with an opticallyactive acid, such as (−)-di-p-toluoyl-d-tartaric acid and/or(+)-di-p-toluoyl-l-tartaric acid followed by fractional crystallizationand regeneration of the free base. The compounds may also resolved byformation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.

[0148] During any of the processes for preparation of the compounds ofthe present invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in Protective Groups in Organic Chemistry, ed. J. F. W.McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, John Wiley & Sons, 1991. The protectinggroups may be removed at a convenient subsequent stage using methodsknown from the art.

[0149] The method of treating conditions modulated by DP I described inthe present invention may also be carried out using a pharmaceuticalcomposition comprising any of the compounds as defined herein and apharmaceutically acceptable carrier. The pharmaceutical composition maycontain between about 0.01 mg and 500 mg, preferably about 5 to 50 mg,of the compound, and may be formulated into any form suitable for themode of administration selected. Carriers include necessary and inertpharmaceutical excipients, including, but not limited to, binders,suspending agents, lubricants, flavorants, sweeteners, preservatives,dyes, and coatings. Compositions suitable for oral administrationinclude solid forms, such as pills, tablets, caplets, capsules (eachincluding immediate release, timed release and sustained releaseformulations), granules, and powders, and liquid forms, such assolutions, syrups, elixirs, emulsions, and suspensions. Forms useful forparenteral administration include sterile solutions, emulsions andsuspensions. Formulations or topical administration include creams,gels, sprays etc.

[0150] Advantageously, compounds of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of transdermal delivery system, thedosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Preferably, the compounds ofthe present invention are administered topically.

[0151] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders; lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbetalactose, corn sweeteners, natural and synthetic gums such as acacia,tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like.

[0152] The liquid forms in suitable flavored suspending or dispersingagents such as the synthetic and natural gums, for example, tragacanth,acacia, methyl-cellulose and the like. For parenteral administration,sterile suspensions and solutions are desired. Isotonic preparationswhich generally contain suitable preservatives are employed whenintravenous administration is desired.

[0153] The compound of the present invention can also be administered inthe form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles, and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, such ascholesterol, stearylamine or phosphatidylcholines.

[0154] Compounds of the present invention may also be delivered by theuse of monoclonal antibodies as individual carriers to which thecompound molecules are coupled. The compounds of the present inventionmay also be coupled with soluble polymers as targetable drug carriers.Such polymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol, olyhydroxyethylaspartamidephenol,or polyethyl eneoxidepolyllysine substituted with palmitoyl residue.Furthermore, the compounds of the present invention may be coupled to aclass of biodegradable polymers useful in achieving controlled releaseof a drug, for example, polyactic acid, polyepsilon caprolactone,polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

[0155] Compounds of this invention may be administered in any of theforegoing compositions and according to dosage regimens established inthe art whenever treatment of the addressed disorders is required.

[0156] The daily dosage of the products may be varied over a wide rangefrom 0.01 to 1.000 mg per adult human per day. For oral administration,the compositions are preferably provided in the form of tabletscontaining, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0,100, 150, 200, 250 and 500 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Aneffective amount of the drug is ordinarily supplied at a dosage level offrom about 0.1 mg to about 500 mg per adult human per day. Preferably,the range is from about 1 to about 250 mg per adult human per day.

[0157] For topical administration, the compositions are preferablyprovided in the form of creams, gels or sprays, containing from about0.01 to 10%, preferably 0,1 to 10%, most preferably 1 to 10% of theactive ingredient.

[0158] Examples of typical creams for topical administration are listedbelow. 1. Basic cream DAC Glycerolmonostearate 60 4.0 g Cetylalcohol 6.0g Mid sized triglycerides 7.5 g Vaseline 25.5 gMacrogel-1000-glycerolmonostearate 7.0 g Propylenglycol 10.0 g Water40.0 g 2. Eucerin cum aqua Lanolin wax alcohol cream 50.0 g Water 50.0 g

[0159] The compounds may be administered on a regimen of 1 to 4 timesper day.

[0160] Optimal dosages to be administered may be readily determined bythose skilled in the art, and will vary with the particular compoundused, the mode of administration, the strength of the preparation, themode of administration, and the advancement of disease condition. Inaddition, factors associated with the particular patient being treated,including patient age, weight, diet and time of administration, willresult in the need to adjust dosages.

[0161] For oral administration, the compounds or compositions of thepresent invention may be taken before a meal, while taking a meal orafter a meal.

[0162] When taken before a meal, the compounds or compositions of thepresent invention can be taken 1 hour, preferably 30 or even 15 or 5minutes before eating.

[0163] When taken while eating, the compounds or compositions of thepresent invention can be mixed into the meal or taken in a separatedosage form as described above.

[0164] When taken after a meal, the compounds and compositions of thepresent invention can be taken 5, 15, or 30 minutes or even 1 hour afterfinishing a meal.

EXAMPLES Example 1 Synthesis of TFA*H-Gly-L-Phe-NHO-Ac (10)

[0165] TFA*H-Gly-L-Phe-NHO-Ac (10) was prepared according to Method D.The compound was purified by flash chromatography to give the product asa white solid (93%) of m.p. 71-73° C.—TLC (n-BuOH/ethylacetate/water/acetic acid, 1:1:1:1): R_(f)=0.73.—¹H NMR (400 MHz,DMSO-d₆): δ=2.16 (s, 3 H, CH₃), 2.80 (dd, 1 H, J=14.0 Hz, J=9.7 Hz, CH₂Phe), 3.04 (dd, 1 H, J=13.9 Hz, J=9.6 Hz, CH₂ Phe), 3.40 (d, 1 H, J=16.3Hz, CH₂ Gly), 3.55 (d, 1 H, J=16.3 Hz, CH₂ Gly), 4.58-4.64 (m, 1 H, CHPhe), 7.19-7.30 (m, 5 H, aryl-H), 8.84 (d, 1 H, J=8.4 Hz, NH).—MS (EI)m/z (%): 280 [M+H⁺].

Example 2 Synthesis of TFA*H-Gly-L-Phe-NHO-Bz (11)

[0166] TFA*H-Gly-L-Phe-NHO-Bz (11) was prepared according to Method D.The compound was purified by flash chromatography to give the product asa white solid (66%) of m.p. 75-80° C.—TLC (n-BuOH/ethylacetate/water/acetic acid, 1:1:1:1): R_(f)=0.81.—¹H NMR (500 MHz,DMSO-d₆): δ=2.86 (dd, 1 H, J=13.9 Hz, J=10.0 Hz, CH₂ Phe), 3.13 (dd, 1H, J=13.9 Hz, J=9.9 Hz, CH₂ Phe), 3.43 (d, 1 H, J=16.3 Hz, CH₂ Gly),3.58 (d, 1 H, J=16.3 Hz, CH₂ Gly), 4.68-4.72 (m, 1 H, CH Phe), 7.21-7.25(m, 1 H, aryl-H), 7.26-7.31 (m, 4 H, aryl-H), 7.58-7.63 (m, 2 H,aryl-H), 7.74-7.77 (m, 1 H, aryl-H), 7.93-8.03 (m, 2 H, aryl-H), 7.94(s, br., 3 H, NH₃ ⁺), 8.91 (d, 1 H, J=8.3 Hz, NH), 12.46 (s, br., 1 H,NH).—MS (EI) m/z (%): 342 [M+H⁺].

Example 3 Synthesis of TFA*H-Gly-L-Phe-NHO-Bz-p-CH₃ (12)

[0167] TFA*H-Gly-L-Phe-NHO-Bz-p-CH₃ (12) was prepared according toMethod D. The compound was purified by flash chromatography to give theproduct as a white solid (82%) of m.p. 98-101° C.—TLC (n-BuOH/ethylacetate/water/acetic acid, 1:1:1:1): R_(f)=0.75.—¹H NMR (400 MHz,DMSO-d₆): δ=2.41 (s, 3 H, CH₃), 2.85 (dd, 1 H, J=13.7 Hz, J=10.0 Hz, CH₂Phe), 3.12 (dd, 1 H, J=12.9 Hz, J=10.0 Hz, CH₂ Phe), 3.42 (d, 1 H,J=15.8 Hz, CH₂ Gly), 3.58 (d, 1 H, J=16.0 Hz, CH₂ Gly), 4.66-4.72 (m, 1H, CH Phe), 7.21-7.25 (m, 1 H, aryl-H), 7.27-7.31 (m, 4 H, aryl-H), 7.40(d, 2 H, J=8.0 Hz, aryl-H), 7.90 (d, 2 H, J=8.2 Hz, aryl-H), 7.94 (s,br., 3 H, NH₃ ⁺), 8.90 (d, 1 H, J=8.4 Hz, NH), 12.40 (s, br., 1 H,NH).—MS (EI) m/z (%): 356 [M+H⁺].

Example 4 Synthesis of TFA*H-Gly-L-Phe-NHO-Bz-p-NO₂ (13)

[0168] TFA*H-Gly-L-Phe-NHO-Bz-p-NO₂ (13) was prepared according toMethod D. The compound was purified by flash chromatography to give theproduct as a white solid (32%) of m.p. 80-85° C.—TLC (n-BuOH/ethylacetate/water/acetic acid, 1:1:1:1): R_(f)=0.77.—¹H NMR (400 MHz,DMSO-d₆): δ=2.86 (dd, 1 H, J=13.9 Hz, J=9.8 Hz, CH₂ Phe), 3.12 (dd, 1 H,J=13.9 Hz, J=9.6 Hz, CH₂ Phe), 3.44 (d, 1 H, J=16.6 Hz, CH₂ Gly), 3.58(d, 1 H, J=16.6 Hz, CH₂ Gly), 4.68-4.74 (m, 1 H, CH Phe), 7.21-7.26 (m,1 H, aryl-H), 7.28-7.32 (m, 4 H, aryl-H), 7.94 (s, br., 3 H, NH₃ ⁺),8.25 (d, 2 H, J=8.9 Hz, aryl-H), 8.39 (d, 2 H, J=8.9 Hz, aryl-H), 8.93(d, 1 H, J=8.4 Hz, NH), 12.69 (s, br., 1 H, NH).—MS (EI) m/z (%): 387[M+H⁺].

Example 5 Synthesis of TFA*H-Sar-L-Phe-NHO-Bz (14)

[0169] TFA*H-Sar-L-Phe-NHO-Bz (14) was prepared according to Method D.The compound was purified by flash chromatography to give the product asa white solid (82%) of m.p. 69-71° C.—TLC (n-BuOH/ethylacetate/water/acetic acid, 1:1:1:1): R_(f)=0.64.—¹H NMR (400 MHz,DMSO-d₆): δ=2.45 (s, 3 H, CH₃), 2.86 (dd, 1 H, J=13.9 Hz, J=10.3 Hz, CH₂Phe), 3.14 (dd, 1 H, J=13.9 Hz, J=10.3 Hz, CH₂ Phe), 3.58 (d, 1 H,J=16.1 Hz, CH₂ Gly), 3.72 (d, 1 H, J=16.1 Hz, CH₂ Gly), 4.69-4.75 (m, 1H, CH Phe), 7.20-7.26 (m, 1 H, aryl-H), 7.28-7.33 (m, 4 H, aryl-H), 7.60(t, 2 H, J=7.8 Hz, aryl-H), 7.76 (t, 1 H, J=6.8 Hz, aryl-H), 8.02 (d, 2H, J=7.0 Hz, aryl-H), 8.63 (s, br., 2 H, NH₂ ⁺), 9.02 (d, 1 H, J=8.6 Hz,NH), 12.48 (s, br., 1 H, NH).—MS (EI) m/z (%): 356 [M+H⁺].

Example 6 Biological Evaluation

[0170] Spectrophotometric Assay:

[0171] DP I was obtained from Qiagen. The DP I activity was determinedin a continous spectrophotometric rate assay using the substrateHCl*Gly-L-Arg-pNA (obtained from Bachem, λ=405 nm) on a HTS 7000 plusmicroplate reader (PerkinElmer, Überlingen, Germany). The assay wasperformed at 30° C., using a MES-buffered system (pH 5.6, 0.104 M(2-[4-Morpholino]ethanesulphonic acid) hydrat/0.0104 M KCl) containing0.0104 M dithiothreitol and 0.0052 M EDTA. The obtained data wereanalyzed with the enzyme kinetic calculation program Grafit 4.016(Erithacus Ltd, UK).

Example 7 Determination of k_(i)-values

[0172] For the K_(i)-value determination a UV-spectrophotometer (Lambda20, PerkinElmer, Überlingen, Germany) was used. The reaction wasmonitored via the p-nitroaniline released from the substrate(Gly-Arg-pNA) at λ=390 nm. DP I (1 U/mg, assay condition: Tagzymehandbook, Qiagen, Hilden, Germany) was diluted in MES-buffer (1:1000)and preincubated and activated for 30 min on crashed ice. The reactionmixture consists: 500 μl stock solution MES-buffer, 500 μlHCl*Gly-L-Arg-pNA, 250 μl inhibitor, 50 μl DP I. In order to calculatethe second order rate constant (k_(inact)/K_(i)) two substrateconcentrations (1 mM and 0.5 mM in the incubation mixture) were combinedwith 6 inhibitor concentrations (5*10⁻⁷ M to 3*10⁻⁸ M in the assay).From the product-time-course the observed first order rate constant wasdetermined.

[0173] $A\overset{k_{obs}}{\rightarrow}P$$v = {{- \frac{\lbrack A\rbrack}{\lbrack t\rbrack}} = {\frac{\lbrack P\rbrack}{\lbrack t\rbrack} = {k_{obs}*\lbrack A\rbrack}}}$

[0174] Plotting 1/k_(obs) versus substrate concentration allows thecalculation of the real first order rate constant of inactivation foreach inhibitor concentration at [S]=0.$k = \frac{k_{inact}*\lbrack I\rbrack}{K_{i} + \lbrack I\rbrack}$

[0175] K_(i) dissociation constant of the enzyme-inhibitor complex

[0176] k_(inact) inactivation rate constant

[0177] A substrate

[0178] P product

[0179] [S] substrate concentration

[0180] [I] inhibitor concentration

[0181] Based on this equation K_(i) and k_(inact) can be determined.

Example 8 Cytotoxicity Assay

[0182] HEP-G2, a human hepatocyte cell line (ACC180) and L-929, a mousefibroplast cell line (ACC2) were grown in RPMI 1604 with 10% fetalbovine serum and 60 μg/ml gentamycin. All cultures were fed every 2-3days and incubated in an humified chamber at 37° C. and 5% CO₂. Fordetermining the cytotoxicity of different DPI-inhibitors, we used theCytoTox96™ Non-radioactive Cytotoxicity Assay (Promega, madison, Wis.,USA). HEP-G2 (30000 cells per well) and L-929 (20000 cells per well)were placed in 96 well plates in RPMI 1604 with 10% fetal bovine serumand grown for 24 hours. The cells were then incubated with variousconcentrations of different DPI-inhibitors for 24 hours in RPMI 1604with 10% fetal bovine serum. One hour after addition of MTS-solution,the reaction was stopped by adding stop-solution of Promega. Theabsorbance was measured at 490 nm with a spectrophotometer. Allexperiments were performed twice simultaneously in three wells.

What is claimed are:
 1. Compounds of the general formula (I)

wherein R is an acyl-residue including a urethane or peptide, or abranched or unbranched C₁-C₉ alkyl chain, a branched or unbranched C₂-C₉alkenyl chain, a branched or unbranched C₂-C₉ alkynyl chain, a C₃-C₉cycloalkyl, C₄-C₉ carbocyclic, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, all of the above residues optionally being substituted, orR is H, the residue AS—AS is a dipeptide or a mimetic thereof, E is O orS, and R′ is a branched or unbranched C₁-C₉ alkyl chain, a branched orunbranched C₂-C₉ alkenyl chain, a branched or unbranched C₂-C₉ alkynylchain, a C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl, C₂-C₉ heterocycloalkyl,C₃-C₉ heterocycloalkenyl, C₅-C₁₄ aryl, C₃-C₉ heteroaryl, C₃-C₉heterocyclic, wherein the heterocycloalkyl, heterocycloalkenyl,heteroaryl, heterocyclic residue can have up to 6 hetero atoms in thering, or R′ is an amino acid or a peptide or a mimetic thereof, all ofthe above residues optionally being substituted, or R′ is H or alkoxy,alkenyloxy, alkynyloxy, carbocyclicoxy, heteroraryloxy, heterocyclicoxy,thioether or a substituted residue thereof or pharmaceuticallyacceptable salts thereof.
 2. Compounds according to claim 1, wherein Ris phenyl or naphthyl optionally mono-, di-, or poly-substituted byC₁-C₆ alkoxy, C₁-C₆ alkyl, C₂-C₆ alkenyloxy, C₂-C₆ alkynyloxy, C₃-C₉heteroaryloxy, C₃-C₉ heterocyclicoxy, C₁-C₆ thioether or a substitutedresidue thereof, NO₂, NH₂, F, Cl, Br, I atoms or groups.
 3. Compoundsaccording to claim 1 or 2, wherein R′ is a phenyl or naphthyl. 4.Compounds according to claim 1 or 2, wherein R′ is

wherein V is N or CH and n=1-6.
 5. Compounds according to claim 1 or 2,wherein R′ is

wherein T¹ is CH or N, W¹, X¹, Y¹ and Z¹ are independently from eachother selected from CH₂, NR², N⁺(R³)₂, O, S, SO, S(R⁴)₂, SO₂, with theproviso that at least two of W¹, X¹, Y¹ and Z¹ are CH₂-groups, R², R³and R⁴ are independently from each other a branched or unbranched C₁-C₉alkyl chain, a branched or unbranched C₂-C₉ alkenyl chain, a branched orunbranched C₂-C₉ alkynyl chain, C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl orH, or pharmaceutically acceptable salts thereof.
 6. Compounds accordingto claim 1 or 2, wherein R′ is

wherein T² is C or N⁺, W², X², Y² and Z² are independently from eachother selected from CH, N, N⁺R⁵ or S⁺R⁶, with the proviso that at leasttwo of W², X², Y², and Z² are CH₂-groups, R⁵ and R⁶ are independentlyfrom each other a branched or unbranched C₁-C₉ alkyl chain, a branchedor unbranched C₂-C₉ alkenyl chain, a branched or unbranched C₂-C₉alkynyl chain, C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl or H, orpharmaceutically acceptable salts thereof.
 7. Compounds according toclaim 1 or 2, wherein R′ is

wherein T³, W³, X³, Y³ and Z³ are independently from each other areselected from CH, N⁺R⁷ or S⁺R⁸, with the proviso that at least two ofW³, X³, Y³, and Z³ are CH₂-groups, R⁷ and R⁸ are independently from eachother a branched or unbranched C₁-C₉ alkyl chain, a branched orunbranched C₂-C₉ alkenyl chain, a branched or unbranched C₂-C₉ alkynylchain, C₃-C₉ cycloalkyl, C₄-C₉ cycloalkenyl or H, or pharmaceuticallyacceptable salts thereof.
 8. Compounds according to claim 1 or 2,wherein R′ is

wherein T⁴ is C or N⁺, or pharmaceutically acceptable salts thereof. 9.Compounds according to claim 1 or 2, wherein R′ is an amino acid, apeptide, a dipeptide or a mimetic thereof.
 10. Compounds according toany one of the preceding claims, wherein the residue R′ may be mono-,di-, or poly-substituted by C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ thioether,NO₂, NH₂, F, Cl, Br, I atoms or groups, except when R′ is H. 11.Compounds according to any one of the preceding claims, characterised inthat they are in the form of prodrugs.
 12. A pharmaceutical composition,characterised in that it contains a compound according to any one of thepreceding claims optionally in combination with pharmaceuticallyacceptable carriers and/or adjuncts.
 13. Use of compounds orpharmaceutical compositions according to any one of claims 1 to 12 forthe preparation of a medicament for the treatment of diseases of mammalsby modulation of the activity of DPI and/or DP I-like enzymes of amammal.
 14. The use according to claim 13 for the inhibition of DP Iand/or DP I-like enzymes.
 15. The use according to claim 14 for thetreatment of malignant cell degeneration, immune diseases and metabolicdiseases of humans.
 16. The use according to claim 14 for improving thewound-healing process and for the treatment of impaired wound-healing inhumans.